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Introduction

Cholera has historically been occurred in periodic epidemic form limited to few developing and under developing countries, namely Bangladesh, India, countries in Africa and South America.V.choleraeis known to be an autochthonousinhabitant of brackish water and estuarine system. Among more than 200 serogroups ofV.choleraeso far identified,¹only O1 and O139 have caused epidemic cholera.^2-5The other serogroups ofV.choleraecollectively referred to as non-O1 andnon-O139 serogroup have not been associated with epidemic but can cause sporadic diarrhoea⁶and ubiquitously distributed in aquatic environment.⁷More than 95% of the strains belonging to serogroups of O1 and O139 produce cholera toxin (CT) which is central to the disease process are designated as toxigenicV.cholerae.

It was believed thatV. choleraeO1 and O139 are able to survive only a few hours in aquatic environment.⁸But later this idea was changed with the observation that the presence of the microorganisms in aquatic environment does not solely dependent on the extent of fecal contamination, as there is no correlation between the presence of fecal coliform bacteria and toxigenic and non toxigenic strains ofV.choleraeO1 and O139 in aquatic environment.⁹Subsequent hypothesis reported toxigenicV.choleraeis an autochthonous member of the microbial flora found in brackish waters typical of estuaries and coastal swamps and can be detected for extended periods in fresh waters, where there is no human fecal contamination.¹⁰

In response to environmental stress in aquatic environments such as low concentrations of nutrients and low temperatures,V.choleraeO1, O139 and Non-O1 and Non-O139 adopt a viable state that enables them to carry out metabolic functions and form colonies but can not be expressedin vitroculture.¹¹If favorable environmental condition prevailsV.choleraecan become culturable again.V.choleraeO1 & O139 in a viable but non-culturable state has produced clinical symptoms of cholera in volunteers which confirms that it maintains its pathogenicity in aquatic environment despite the inability of the cells to be cultured.¹¹Endemic and seasonal nature of cholera depends on the presence of the pathogenic agents in a viable but non-culturable state in an aquatic ecological niche that serves as reservoirs for the agent between the epidemic periods.¹¹The objective of this article is to focus the ecological interaction of toxigenicV.choleraeassociated with biotic and abioticfactors, and its effect on survival and genome ofV. choleraein aquatic environment.

It is well understood that to control cholera it is necessary to prevent humans from coming into contact with the natural reservoirs of toxigenicV.cholerae. This implies the ability to identify aquaticenvironmental abiotic and biotic ecological conditions that enable the microorganisms to survive between epidemics. During this period,V.choleraemay adopt different conditions to acclimatize the adverse situation which may lead to changes in genetic make up. To determine which aquatic ecosystem can harbor the microorganisms where many phenotypic and genotypic character changes occur it is essential to understand the ecology ofV.choleraewhich will contribute to understand theendemic nature of this disease.

Ecology of ToxigenicVibrio Cholerae水

V.choleraeincluding its toxigenic strains¹²has often been isolated from aquatic environments such as bays, rivers, canals, ponds, ditches and ground water. Cholera transmission takes place primarily through ingestion of water contamination with the feces or vomitus of patients or less frequently with the feces of asymptomatic carriers. Both the toxigenicV.choleraeO1和O139已经从水生enviro分离nments and are believed to spread primarily by water.^13-14

Nutrients

V.choleraeis facultative anaerobes thatcan grow in media containing carbohydrates, nitrogen, sulfur, phosphorous and sodium; to obtain such minerals it adheres to sediments. ToxigenicV.choleraeO1和O139需要Na⁺to survive inabsence of nutrients. In presence of Na⁺, addition of alkaline earth metals Ca⁺and Mg⁺, can prolong the survival period ofV. cholerae¹⁵. Iron plays a significant role in life process ofV.choleraewhich is absorbed after being chilated by siderspore, vibriobactin.

Salinity

In absence of nutrients, the ideal salinity for growth of toxigenicV.choleraeis 25 parts per 1000V.choleraecan grow in aquatic environment of high salinity (45 parts per 1000) if it receives 500µg or more tryptone as a substrate. HoweverV.choleraeis able to survive for extended periodsand can multiply in fresh water environments in the presence of adequate concentration of nutrients which can meet the minimum need of salinity.¹⁶

Temperature

The ideal growth temperature for toxigenicV.choleraevaries between 30⁰and 37⁰C. ToxigenicV.choleraecan survive for prolonged period insummer than in the winter.¹⁰

Acidity

ToxigenicV.choleraeO1 and O139 can tolerate alkaline environments and is very sensitive to acidity.¹⁵The optimal pH for survival in 25⁰C water is between 7 and 8.5 when salinity is moderate and between 7.5 and 9 when salinity is low.

Biotic Factors

Aquatic Macrophytes

ToxigenicV.choleraein fresh water aquatic environment adheres to the roots of macrophytes likeEichhornia crassipes(water hyacinths) which favors its survival. The pathogenic agent secretes the enzyme mucinase, which is considered to be one of the factors responsible for the virulence of theV.choleraeand which degrades the cellular mucilage of plants.¹⁷Aquatic plants could be environmental reservoirs of the microbes either through a non-specific association or a commensal relationship.¹⁷

Phytoplankton

V.choleraeserogroups target species ofsea and fresh water phytoplankton and zooplankton to which it adheres.V.choleraeprimarily colonizes the oral regions and egg sacks of the planktonic copepods. Reproduction ofV.choleraetakes place in egg sacks, the digestive system and the chitinous exoskeleton of copepods.¹⁸The pathogenic agents secrete chitinase, an enzyme that enables it to digest chitin and use it as source of nutrients. Oviposition and expulsion of fecal material by planktonic copepods can expedite dissemination and reproduction of the pathogenic microorganisms in aquatic environments.¹⁸ToxigenicV.choleraeO1 strain can attach the species of green algae and blue green algae that can survive longer due to its ability to derive nutrients from the extra cellular products released by these species.¹⁹

Viable but non-culturable Vibrio mostly found in aquatic environment during the inter-epidemic period due to nutritional defficiency. Culturable Vibrio in aquatic environment adheres to the plankton which withstands the seasonal changes in temperature, salinity, pH and nutrient concentration and that they enter a non-culturable state for a given period as a way to adapt to any adverse situation. Once favorable growth conditions return,V.choleraeagain adapt its culturable state and pose threat of an epidemic if certain plankton blooms contribute to its reproduction.

Fish, Mollusks and Crustaceans

In some geographical region,V.choleraehas been isolated from shrimps and crabs as well as from Oysters and the intestines of fish. The chitinous surface of the crustaceans provides a suitable substrate for reproduction of the pathogenicity microbes. There has been evidence of existence of an association between the incidence of cholera and the consumption of fish and other raw or undercooked seafood.²⁰

Aquatic Birds

V.choleraecolonizes in certain great blueherons which were detected in its feces but not from water samples collected from bird’s habitat. Such aquatic birds can be the carriers of the pathogenic agent and contribute to its overall dissemination.²¹

Seasonality

During inter-epidemic periodV.choleraepasses its lifecycle in aquatic ecosystem closely associated with the micro-niches. The density ofV.choleraevaries seasonally depending on thesignificant influence of temperature in aquatic environment.²²In aquatic ecosystemV.choleraetake advantage of micro-niches for the perpetuation of its lifecycle. Population densities ofVibrio choleraeare ultimately determined by the adequate presence of niches which in turn is closely triggered by the temperature. In winter season the count ofV.choleraeremain below detectable levels inplankton and in water sample. Water sediment harbors highest densities ofV.choleraeimmediately after the months when peak appearance is observed in plankton. This suggests the maintenance of lifecycle ofV.choleraebetween the niches with respect to the season.²³

The Clinical and Environmental Divide

After shedding from the intestinal environment of human host, ifV.choleraereaches aquatic environment passes its life cycle at different state depending on the types of niches in water under various conditions of temperature, salinity, pH, nutrients and environmental stress. Due to the long term starvation, the loss of toxin producing ability of toxigenicV.choleraewas observed in aquatic ecosystem²⁴which may be due to natural stress. AlthoughV .choleraeO1 has isolated frequently from aquatic environs most of the O1 strains do not produce cholera toxins. However some recent studies have reported the presence of ofctxAB gene, encoding cholera toxin in environmentalV. cholereaeO1 and O139 strains.^25,14During the prolong starvation,V.choleraelosses its external morphology of curved rod shaped bacilli and coverts to cocci shape besides the change of physiology of metabolic process. However upon exposure to the favorable condition like intestinal milieu,V.choleraerestores the capacity to express the phenotyping and genotyping character.

Genomic Profiles and Environment Interaction

Several examples of microevolution are known to exist in the context ofV.cholerae. Horizontal gene transfer of O antigen genes has been shown to occur in the generation of O139 and O37 serogroups and variability in thetcpAgene is believed to be caused by homologous recombination.²⁶Rapid microevolution occurs amongV.choleraestrains and that gene flow is not restricted or bottlenecked between environmental and clinical habitats.²⁷EnvironmentV.choleraepopulation demonstrates significant geographical isolation but barriers between the clinical habitats and aquatic environment are not significant. In addition to spatial variance, temporal variance is a significant factor explaining total genomic variances among toxigenicV.choleraepopulation.²⁷Aquatic environment being the reservoir of toxigenicV.cholerae, dynamics of its population contributesignificantly to variation in cholera epidemics. Any change in the compositionV.choleraepopulation in the aquatic environment that may be driven by seasonal fluctuation in the environment or by introduction of new strains through microevolution or being imported from other system can cause coupled changes in composition and behavior of the clinical population leading to a shift in the dynamics of disease expression in cholera.

Clonal Shift

Clonal diversity and continual emergence of new epidemic clone among toxigenicV.choleraehas been evidenced by molecular epidemiological studies. Cholera toxin which is responsible for profuse watery diarrhoea is encoded by a lysogenic bacteriophage designated as CTXphi. CTXphi plays an important role for the emergence of new toxigenic clone ofV.cholerae.The ecosystem comprising ofV.cholerae, CTXphi, the aquatic environment, andmammalian host offers an understanding of the complex relationship between pathogenesis and the natural selection of a pathogen. Changes in survival capacity ofV.choleraecombating intestine immunity or stresses in environmental habitats is another potential factor for the institution of clonal shift as has been witnessed the wide spread occurrences of El Tor vibrio in environment of south Africa and Latin America.²⁸

In general, it is impossible to separate environment factors from biological factors, as can be seen from interrelationships in nature that plays a significant role in the emergence of infectious diseases. In conclusion,V. choleraean environmental inhabitant of brackish, estuarine and marine ecosystem represents an agent of disease that can be dramatically influenced by environmental changes including global environmental changes.

Acknowledgements

The authors are thankfully acknowledged to Regional Medical Research Centre, Bhubaneswar (ICMR) for preparation of the manuscript.

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